Survival in ovarian cancer in Wales: Prior to introduction of all Wales guidelines.

The objective of the study was to review referral practice, overall management, and survival in women with suspected ovarian cancer in Wales. This study was done prior to introduction of cancer management guidelines in the region. A confidential study questionnaire was sent to 20 participating hospitals. Data on 287 consecutive women with suspected ovarian cancer were collected, of which 250 women underwent primary laparotomy. Information was obtained on referral pattern, preoperative investigations, place of primary surgery, specialty of the primary surgeon, surgical parameters recorded at the time of operation, a final overall stage, adjuvant treatment, and survival outcome. There was a wide variation in referral practice and management of ovarian cancer in Wales. Stage of the disease, attempt at optimal debulking, residual disease, management by a cancer centre multidisciplinary team, and platinum-based chemotherapy were associated with improved overall survival and progression-free survival. More women were alive if managed in the cancer centre at 1 and 3 year after diagnosis (P = 0.022). This study has highlighted the acute issue of the standards of clinical care in the area of ovarian cancer management and will emphasize the implementation of better care pathways for ovarian cancers.

Association between glutathione-S-transferase GSTP1 genotypes, GSTP1 over-expression, and outcome in epithelial ovarian cancer.

Ovarian cancer accounts for the majority of deaths from gynaecological malignancy, and polymorphisms in genes encoding the glutathione-S-transferase (GST) GSTP1 detoxifying enzymes may lead to variation in detoxification of carcinogens. We describe a study involving 81 women with invasive epithelial ovarian cancer. A number of important clinical variables and outcome data were obtained. GSTP1 genotyping was undertaken using PCR-based techniques, and GSTP1 expression was quantified using immunohistochemistry (IHC). A Cox's proportional hazard regression model was used to analyze the effects on outcome. We also independently examined 11 women with borderline or low malignant potential (LMP) tumors using IHC only. The mean age of the women was 61.5 years +/- 12 (1 SD) (range 36-88 years), the median overall survival was 26 months, and median progression free interval (PFI) 21 months. There was a significant association between GSTP1 (Val(104)/Val(104)) genotypes, and reduced survival (P = 0.05) and the GTP1 (Ile(104)/Val(104)) genotype appeared to have the best outcome (HR = 0.34, P = 0.045, 95% CI = 0.12-0.98). There was no significant association between the GSTP1 genotypes and any clinico-pathological parameters; there were also no associations between GSTP1 genotypes and response to postoperative chemotherapy. Specific nuclear GSTP1 over-expression was associated with less residual disease (P = 0.05); specific cytoplasmic GSTP1 over-expression with more favourable performance status (P = 0.014)). We found that 10/11 (91%) of the LMP (borderline) tumors over-expressed nuclear GSTP1 compared to only 52% of the invasive tumors (chi(2) ((1)) = 5.95, P = 0.015). There was no significant association between the level of GSTP1 expression and response to postoperative chemotherapy. The overall level of GSTP1 expression and the subcellular localization of GSTP1 expression were not associated with either survival or PFI. There was a significant association between the GSTP1 (Ile(104)/Ile(104)) genotypes and increased overall GSTP1 expression (P = 0.049), and the GSTP1 (Ile(104)/Val(104)) genotypes and reduced overall GSTP1 expression (P = 0.046). We speculate that GSTP1 Ile(104)/Val(104) genotypes are associated with improved outcome because the protein/enzyme, which is expressed, may provide a better balance between the effects of detoxification of carcinogens and the effects of metabolism of chemotherapy agents. In addition, over-expression of nuclear GSTP1 appears to be associated with more favorable ovarian tumor characteristics. In our preliminary study, we also reported a relationship between overall GSTP1 expression and certain GSTP1 genotypes. As far as we are aware, this is the first time that a relationship between the GSTP1 genotypes, GSTP1 expression and outcome has been described in ovarian cancer. Whether the genotype directly determines GSTP1 expression is at present unclear and the precise mechanism of this interaction is unknown.

The role of clinical follow up in early stage cervical cancer in South Wales.

OBJECTIVE: To assess the effectiveness of clinical follow up after primary surgery for early stage cervical cancer. DESIGN: Retrospective analysis of clinical follow up after radical hysterectomy and node dissection for early stage cervical cancer. SETTING: Gynaecological Oncology Cancer Centre. SAMPLE: Two hundred and ninety-one patients who underwent surgery for cervical cancer. METHODS: Follow up data were collected retrospectively from hand-searched patients notes, as well as a computerised database (Information System for Clinical Organisation [ISCO]). The data were analysed using the SPSS for windows (SPSS, Chicago, Illinois) statistics package, using chi2, Kaplan-Meier life tables and Cox Linear regression analysis. MAIN OUTCOME MEASURES: To determine whether routine follow up was useful for detecting early recurrent disease. RESULTS: Two hundred and ninety-one patients treated by radical hysterectomy and node dissections were followed up. The cumulative five-year survival for all cases in our series was 80% and 53/291 patients (18.2%) were found to have recurrent disease. The median period from surgery to recurrence was 17.6 months (3.0-60.0). Seven patients with recurrence were detected at a routine follow up examination, and two out of seven of the patients were asymptomatic. Detection of the recurrence on routine follow up was not an independent prognostic factor for survival when compared with age, stage and whether the patient received post-operative adjuvant therapy. CONCLUSIONS: Routine follow up in patients following radical hysterectomy and node dissection for early stage cervical cancer is not a sensitive way of detecting recurrent disease, as a high proportion of patients were symptomatic at the time of detection. As there are other reasons for follow up, we propose alternative methods of structuring the programme.

Follow-up after negative large loop excision of the transformation zone (LLETZ) of the cervix.

We performed a retrospective analysis of 204 patients who underwent large loop excision of the transformation zone (LLETZ) to determine whether women who have a negative LLETZ (no cervical intra-epithelial neoplasia (CIN) present) are at a lower risk of developing further abnormal cytology or CIN than women whose LLETZ is positive (CIN present). Overall 69 of the LLETZ samples were negative (34%) and 135 were positive (66%). The mean duration of negative cytology during follow up was 25.4 months (negative LLETZ) and 21.2 months (positive LLETZ) (P=0.03). In the negative LLETZ group, cytology did not miss any cases of persistent CIN at the 6-month follow-up visit and 39 repeat punch biopsies were all negative. There were 3/69 cases (4.3%) in the negative LLETZ group and 13/135 (9.6%) in the positive LLETZ group of persistent disease (CIN diagnosed at or as a result of the 6-month follow-up). Following a negative 6-month review, one woman developed an abnormal smear in the negative LLETZ group (1/66=1.5%) compared with 10 women (10/122=8.1%) in the positive LLETZ group. Recurrent CIN (CIN subsequently diagnosed following a negative 6-month review) was seen in 0/69 cases (0%) in the negative LLETZ group and 4/135 (3.2%) in the positive LLETZ group. We conclude that women who undergo a negative LLETZ may represent a low-risk group for developing further cytological and histological abnormalities.

Glutathione S-transferase GSTM1 and GSTT1 genotypes in ovarian cancer: association with p53 expression and survival.

The objective of this study was to determine whether the association between GSTM1 null/GSTTI null and survival in ovarian cancer is mediated by the influence of these genes on p53 expression. In 81 women with pure invasive ovarian cancer, GSTM1 null and GSTT1 null genotypes were identified using polymerase chain reaction and p53 expression was assessed using immunohistochemistry. The association of these factors with survival was examined using Cox's proportional hazards regression models. Performance status (P < 0.001), operative stage (P = 0.004), residual disease (P = 0.001), histologic subtype (P = 0.05), tumor grade (P = 0.007), and the combined GSTMI null/GSTTl null genotype (P = 0.023) were all individually associated with survival. p53 expression was not associated with survival (P = 0.45). In a multivariate analysis, the effects of GSTM1 null/GSTT1 null on survival were lost when residual disease and tumor grade were included. The effects of p53 expression on survival were unchanged when residual disease, tumor grade, operative stage, and performance score were included. GSTM1 null/GSTT1null did not influence the effects of p53 expression on survival and vice versa. The GSTM1 null/GSTT1 null genotype was associated with response to primary chemotherapy (P = 0.007) but p53 expression was not. We conclude that the association of GSTM1 null/GSTTl null with survival appears to be mediated through different mechanisms to p53 expression in ovarian cancer and in addition, may be a better predictor of outcome.

How can the incidence of negative specimens resulting from large loop excision of the cervical transformation zone (LLETZ) be reduced? An analysis of negative LLETZ specimens and development of a predictive model.

OBJECTIVE: To analyse biopsies of large loop excision of the transformation zone of the cervix; to identify factors associated with negative histology; and to develop predictive models in order to reduce the number of negative loop excisions. DESIGN: Retrospective analysis of patient notes and audit database. SETTING: Colposcopy clinic of a large district general hospital in North Staffordshire. POPULATION: Four hundred and fifty-two women who underwent a large loop excision of the transformation zone (LLETZ) procedure for suspected cervical intraepithelial neoplasia. METHODS: Women who underwent a LLETZ procedure were placed in two different groups, one positive for cervical intra epithelial neoplasia and the other negative for cervical intra epithelial neoplasia. Information was obtained on a number of clinical and colposcopic variables. Analysis was undertaken to determine if there were any differences between the two groups. These factors were then identified and three predictive models generated. Receiver-operator characteristic curves were used to assess and test these models. MAIN OUTCOMES MEASURES: To identify factors associated with negative histology on a LLETZ specimen. To predict how to reduce the number of negative LLETZ specimens. RESULTS: Four hundred and fifty-two women underwent a LLETZ procedure, 88 were negative (19%) and 364 were positive (81%). In women who were treated at their first visit, 56/316 (18%) had negative histology. There were significant associations between negative histology in the LLETZ and negative or low grade cytological atypia, negative colposcopic findings and years of age > 50 in both bivariate analysis and stepwise logistic regression. In the predictive models, the sensitivity ranged between 72% and 80%, the specificity 59%-72%, and the area under the receiver-operator characteristic was 0.75-0.77. If we had used the predictor models and managed women with negative or low grade cervical atypia and negative colposcopy findings conservatively, we would have reduced the negative biopsy rate from 19% to 14%, but five cases of high grade disease and 25 cases of low grade disease would have been missed. If we had also included women aged > 50 years in this model, the negative biopsy rate would have dropped from 19% to 15%, with only one case of high grade disease and 11 cases of low grade disease missed. All these women would require continued cytological and colposcopic surveillance. Importantly, no cases of invasion would have been missed. CONCLUSION: Using a predictive model can reduce the number of negative LLETZ specimens, but at the expense of continued cytological and colposcopic surveillance and cannot be recommended in normal practice. This raises the question whether current standards for negative histology in LLETZ specimens are set unrealistically high.

A comparison of the side effects of prilocaine with felypressin and lignocaine with adrenaline in large loop excision of the transformation zone of the cervix: results of a randomised trial.

OBJECTIVE: To test the hypothesis that prilocaine with felypressin causes fewer side effects than lignocaine with adrenaline when performing large loop excision of the transformation zone of the cervix. DESIGN: Randomised trial. SETTING: Colposcopy clinic in a large district general hospital. PARTICIPANTS: Two hundred consecutive women undergoing large loop excision of the transformation zone of the cervix. METHODS: Two different local anaesthetic combinations (prilocaine with felypressin and lignocaine with adrenaline) were compared in women undergoing large loop excision of the transformation zone. Prospective collection of clinical and treatment data was undertaken with scoring using an ordinal scale of pain experienced by the women during the procedure. Peri-operative blood loss and any side effects were also recorded. MAIN OUTCOME MEASURES: Side effects associated with the local anaesthetic agents. RESULTS: Lignocaine with adrenaline resulted in less blood loss (P = 0.006) but was more likely to cause side effects, such as feeling faint (P = 0.017) and shaking (P < 0.001). CONCLUSION: Prilocaine with felypressin causes fewer side effects than lignocaine with adrenaline and is therefore the preferred local anaesthetic combination for large loop excision of the transformation zone.

Expression and subcellular localization of cyclin D1 protein in epithelial ovarian tumour cells.

The expression of cyclin D1 protein in tumour sections from 81 patients with epithelial ovarian cancer was analysed using immunohistochemistry. The tumours that overexpressed cyclin D1 in more than 10% of neoplastic cells were considered positive. Thus overexpression of cyclin D1 was observed in 72/81 (89%) of the cases examined. Protein was detected in both the nucleus and the cytoplasm in 24/81 (30%) and localized exclusively in the cytoplasm in 48/81 (59%) of the tumours. Cyclin D1 was overexpressed in both borderline and invasive tumours. There was no association between protein overexpression and tumour stage and differentiation. Furthermore, no correlation between cyclin D1 expression and clinical outcome was observed. However, in tumours overexpressing cyclin D1 (n = 72), the proportion displaying exclusively cytoplasmic localization of protein was higher in those with serous compared with non-serous histology (P = 0.004, odds ratio 4.8, 95% confidence interval 1.4-19.1). Western analysis using a monoclonal antibody to cyclin D1 identified a 36 kDa protein in homogenates from seven tumours displaying cytoplasmic only and one tumour demonstrating both nuclear and cytoplasmic immunostaining. Using restriction fragment length polymorphism polymerase chain reaction and PCR-multiplex analysis, amplification of the cyclin D1 gene (CCND1 was detected in 1/29 of the tumours demonstrating overexpression of cyclin D1 protein. We conclude that deregulation of CCND1 expression leading to both cytoplasmic and nuclear protein localization is a frequent event in ovarian cancer and occurs mainly in the absence of gene amplification.

Is the provision of information leaflets before colposcopy beneficial? A prospective randomised study.

OBJECTIVE: To assess the usefulness of a leaflet distributed to women before colposcopy designed to reduce their anxiety and psychosexual morbidity by providing information. DESIGN: Prospective randomised study. SETTING: Colposcopy clinic of a large district general hospital. SAMPLE: Two hundred consecutive women undergoing colposcopy for the first time for a cervical cytological abnormality of severity no greater than moderate dyskaryosis. METHODS: Women were randomised into one of two groups (leaflet or control). Those in the leaflet group were sent an information leaflet prior to attending the clinic. In the colposcopy clinic all the women completed a State/Trait Anxiety Inventory (StAI/TrAI) and a modified psychosexual questionnaire before undergoing colposcopy. This was repeated at the six-month follow up visit. Women in the leaflet group also completed a further questionnaire on the leaflet. MAIN OUTCOME MEASURES: Differences of anxiety and psychosexual scores between leaflet and control groups. RESULTS: The leaflet was well received. There were no statistical differences in StAI and TrAI scores between the study group and the control group at either visit, although in the whole study population StAI and TrAI scores were reduced at the second visit. The leaflet group had significantly more psychosexual problems but by the second visit, the scores had improved and the two groups were similar. When the mean differences in anxiety and psychosexual scores at the initial and second visits were compared between the groups, the reduction in negative sexual feelings and deterioration of TrAI scores experienced by the leaflet group was significant. CONCLUSIONS: This study suggests that the provision of sending an information leaflet prior to colposcopy is not beneficial in isolation. Other approaches need to be considered.

Prognostic significance of cyclin D1 gene (CCND1) polymorphism in epithelial ovarian cancer.

We have investigated the influence of CCND1 genotype on clinical outcome in 138 women with epithelial ovarian cancer. CCND1 genotypes were identified from peripheral blood DNA by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. Patient CCND1 genotypes were compared with clinical details including FIGO tumor stage, residual tumor volume, tumor histology and differentiation, response to chemotherapy, progression free interval, and survival. We observed no association between patient CCND1 genotypes and tumor characteristics or response to chemotherapy. There was no significant difference in overall survival and progression free interval (PFI) among women with different CCND1 genotypes. However, analysis of data from patients who responded to postoperative chemotherapy revealed that women with CCND1 AA genotype were associated with early disease progression (P = 0.020, HR 4.58, 95% CI 1.27-16.48) and reduced survival (P = 0.026, HR 4.48, 95% CI 1.19-16.79) compared with those with CCND1 AG and GG genotypes. These data show that CCND1 genotype does not influence overall prognosis in a cohort of epithelial ovarian cancer patients, however, it is associated with disease progression in a subgroup of patients following initial response to chemotherapy.

Do women referred for colposcopy receive adequate information from the primary care team?

The objective of this study was to assess whether women had been counselled by their primary health care team and whether or not they had received any information about colposcopy. We also asked where the best information had been obtained. The setting was the colposcopy clinic of a large district general hospital, the design of which was a cross-sectional audit using a questionnaire. The subjects were 100 women attending the colposcopy clinic. The results show that 63% of women were seen by their primary health care team before the colposcopy visit and that counselling was associated with knowledge about colposcopy (P = 0.017). However, 43% of women felt that they knew nothing about colposcopy whilst 19% of women thought that the information they had obtained was not useful. We conclude that there is scope for improving the quality, timing and provision of information for women undergoing colposcopy.

Audit of the views of gynaecological cancer patients regarding follow-up.

The purpose of this study was to audit patients opinions on routine follow-up and discharge from the gynaecology clinic. The setting was the gynaecology clinic of a large district general hospital and the design was a cross-sectional audit analysis using a written questionnaire. The subjects were 103 women attending the gynaecological out patients for review following previous treatment for various gynaecological cancers. Ninety patients took part in the study, of these 36 (40%) wished for 'free access', 29 (32%) wished for routine hospital follow-up, and 25 (28%) wished for discharge but with follow-up by their general practitioner. There were no statistically significant differences in choices between cancer groups. The choice of 'free access'seemed to be an important choice for women. We conclude that routine hospital follow-up of women treated for gynaecological cancer is not the desire of the majority and it appears the women may have different inherent psychological characteristics.

Association of glutathione S-transferase GSTM1 and GSTT1 null genotypes with clinical outcome in epithelial ovarian cancer.

Epithelial ovarian cancer is generally associated with a poor outcome, although the mechanisms that determine survival and progression-free interval (PFI) are unclear. Data from ovarian tumors showing associations between (a) null genotypes at the glutathione S-transferase GSTM1 and GSTT1 loci and expression of p53 protein and (b) outcome and expression of p53 suggest that polymorphism at these loci is a factor determining outcome. Accordingly, we have studied the association between the GSTM1 null and GSTT1 null genotypes and survival and PFI in 148 women with epithelial ovarian cancer. Although we did not find an association between individual genotypes and outcome, women with both GSTM1 null and GSTT1 null genotypes demonstrated poorer survival (P = 0.001) and reduced PFI (P = 0.003). Thus, no cases with both these genotypes survived past 42 months postdiagnosis. In contrast, 43% of the women without this combination survived beyond this time. Because response to chemotherapy is a major factor determining outcome in ovarian cancer, we also examined the data for associations between the glutathione S-transferase genotypes and response to such treatment. Thus, in 78 patients treated with chemotherapy, the combination of GSTM1 null and GSTT1 null was associated with unresponsiveness to primary chemotherapy (P = 0.004); none of the eight patients with both these genotypes responded, compared with 38 of 70 (54%) of patients with other genotype combinations. The effect of the combination of genotypes on survival and PFI was lost in a multivariate model that included response to chemotherapy as a confounding factor. This suggests that the combination of GSTM1 null/GSTT1 null is associated with outcome because of its influence on response to chemotherapy. These preliminary findings may provide a basis for the selection of patients for treatment with chemotherapeutic agents.

Relationship of global chloroquine transport and reversal of resistance in Plasmodium falciparum.

Control of falciparum malaria has become almost impossible in many areas due to the development of resistance to chloroquine and other antimalarial drugs. Verapamil and a number of unrelated compounds which chemosensitise multi-drug resistant cancer cells also enhance chloroquine susceptibility in Plasmodium falciparum. Chloroquine is accumulated to lower levels in resistant plasmodia, hence the reversal of chloroquine resistance has been attributed to the ability of chemosensitising agents to increase the amount of chloroquine accumulated by the resistant parasite. We have conducted a detailed examination of the effect of verapamil on chloroquine sensitivity and its relationship to chloroquine accumulation. The ability of verapamil to increase steady-state chloroquine accumulation was found to be totally insufficient to explain the increase in chloroquine sensitivity caused by the drug. In contrast, when chloroquine accumulation was increased by raising the pH gradient, the corresponding shifts in sensitivity to chloroquine could be accurately predicted. These results were confirmed with other classes of chemosensitisers and we conclude that an alternative mechanistic explanation is required to completely explain the reversal of chloroquine resistance in P. falciparum.

The effect of invertebrate hormones and potential hormone inhibitors on the third larval moult of the filarial nematode, Dirofilaria immitis, in vitro.

The effects of the insect hormones, ecdysone and 20-hydroxyecdysone, certain non-steroidal ecdysteroid agonists (RH compounds) and the inhibitor, azadirachtin, on the timing of the 3rd-stage moult of Dirofilaria immitis were investigated. 20-Hydroxyecdysone and RH 5849 when used at a concentration of 10(-5) M, resulted in a premature timing of this moult. Azadiracthin, at a similar concentration, prevented moulting of most of the larvae to the 4th stage. The results are discussed in relation to the possibility of a hormonal role for ecdysteroids and neuropeptide-like compounds in the control of ecdysis in filarial nematodes, that maybe somewhat comparable to the system which is found in insects.

Development of halofantrine resistance and determination of cross-resistance patterns in Plasmodium falciparum.

Intermittent exposure to halofantrine (HF) of both chloroquine-susceptible (T9.96) and chloroquine-resistant (K1) isolates of Plasmodium falciparum in vitro resulted in a rapid reduction in susceptibility to HF. After 6 months, the 50% inhibitory concentration (IC50) of HF, determined with [G-3H]hypoxanthine incorporation as a marker, increased ninefold for the chloroquine-resistant (K1) isolate and threefold for the cloned chloroquine-susceptible (T9.96) isolate, the derived isolates being termed the K1HF3 and T9.96HF4 isolates, respectively. By microscopic examination of cultured erythrocytes, we determined that there was a fivefold increase in the IC50 for isolate T9.96HF4. The responses of the parental isolates and the HF-resistant isolates to chloroquine, mefloquine, quinine, amodiaquine, qinghaosu, and pyrimethamine were determined. In comparison with the parental K1 isolate, HF-resistant isolate K1HF3 was significantly more susceptible to the action of chloroquine and exhibited a significantly reduced susceptibility to quinine and mefloquine. The other HF-resistant isolate, T9.96HF4, showed no alteration in susceptibility to amodiaquine or chloroquine but a significantly decreased susceptibility to mefloquine. Resistance was stable in the two isolates, both in the absence of drug pressure or when kept frozen in liquid nitrogen. In contrast, continuous exposure to HF had no effect on the susceptibility of the parasites to this drug above HF concentrations of 3.2 x 10(-9) M.

Enhancement of drug susceptibility in Plasmodium falciparum in vitro and Plasmodium berghei in vivo by mixed-function oxidase inhibitors.

A number of compounds, as exemplified by verapamil and desipramine, have been shown to enhance the susceptibility of resistant malaria parasites to chloroquine. The mechanism by which these agents reverse resistance is still controversial but is though to involve alterations in drug transport causing an increase in steady-state drug concentrations. We have proposed that an alternative resistance mechanism may involve the metabolic deactivation of the drug in some resistant parasites via cytochrome P-450 mixed-function oxidases. If the hypothesis is true, it should be possible to alter drug susceptibility in malaria parasites by the use of agents known to inhibit or induce cytochrome P-450 activities. We have assessed the ability of known inhibitors of cytochrome P-450 enzymes (cimetidine, metyrapone, and alpha-naphthoflavone) to enhance chloroquine susceptibility in Plasmodium falciparum culture-adapted and wild-type isolates in vitro and P. berghei in vivo. In all three systems, the inhibitor cimetidine enhanced parasite susceptibility to chloroquine, and this increase in susceptibility was unrelated to changes in chloroquine steady-state concentrations in vitro or to alterations in host pharmacokinetics in vivo. Additionally, the cytochrome P-450 inducer phenobarbital produced slight decreases in P. falciparum drug susceptibility in vitro. We have compared the ability of the cytochrome P-450 inhibitors cimetidine and metyrapone to enhance drug susceptibility with that of verapamil by using wild-type malaria isolates obtained from Cameroon. Verapamil completely reversed resistance, i.e., to below the cutoff point of 70 nM, in all the resistant isolates. Cimetidine enhanced chloroquine susceptibility in 60% of the isolates and reduced 50% inhibitory concentrations by at least 43% in all the resistant isolates. The compounds tested had little or no effect on the 50% inhibitory concentrations for the susceptible isolates. The data support a possible role for detoxification in chloroquine resistance, and even in the absence of such a process we have observed apparent chemosensitization by agents whose common biological feature is the inhibition of cytochrome P-450 enzymes. Additionally, sensitization has been observed in wild-type isolates of P. falciparum obtained form immune residents of an area of endemicity as well as culture-adapted parasites.

Rapid chloroquine efflux phenotype in both chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum. A correlation of chloroquine sensitivity with energy-dependent drug accumulation.

Recent reports suggest that lower levels of chloroquine accumulation in chloroquine-resistant isolates of Plasmodium falciparum are achieved by energy-dependent chloroquine efflux from resistant parasites. In support of this argument, a rapid chloroquine efflux phenotype has been observed in some chloroquine-resistant isolates of P. falciparum. In this study, no relationship was found between chloroquine sensitivity and the rate of [3H]chloroquine efflux from four isolates of P. falciparum with a greater than 10-fold range in sensitivity to chloroquine. All the isolates tested displayed the rapid efflux phenotype, irrespective of sensitivity. However, chloroquine sensitivity of these isolates was correlated with energy-dependent rate of drug accumulation into these parasites. Verapamil and a variety of other compounds reverse chloroquine resistance. The reversal mechanism is assumed to result from competition between verapamil and chloroquine for efflux protein translocation sites, thus causing an increase in steady-state accumulation of chloroquine and hence a return to sensitivity. Verapamil accumulation at a steady-state is increased by chloroquine, possibly indicating competition for efflux of the two substrates. Increases in steady-state verapamil concentrations caused by chloroquine were identical in sensitive and resistant strains, suggesting that similar capacity efflux pumps may exist in these isolates. These data suggest that differences in steady-state chloroquine accumulation seen in these isolates can be attributed to changes in the chloroquine concentrating mechanism rather than the efflux pump. It seems likely that chloroquine resistance generally in P. falciparum, results at least in part from a change in the drug concentrating mechanism and that changes in efflux rates per se are insufficient to explain chloroquine resistance.